1.1 Field of the Invention
The present invention relates generally to the fields of genetics and cellular biology. More particularly, it concerns a yeast based system for the determination of compounds that affect amyloid formation. The present invention relates to the determination of compounds that affect the amyloid associated with Alzheimer's disease, Transmissible spongiform encephalopathies (TSEs), and several rare human neuropathies: Creutzfeld-Jacob disease (CJD), fatal familial insomnia (FFI), Gertsmann-Straussler-Scheinker (GSS) syndrome, and kuru.
1.2 Description of Related Art
1.2.1 Yeast Prions
Recently, a novel mode of inheritance has been discovered in Saccharomyces cerevisiae (Wickner, 1994; Lindquist, 1997). Phenotypes transmitted by two dominant, cytoplasmically inherited genetic elements, [PSI+] and [URE3], seem to depend upon the inheritance of altered protein structures, rather than altered nucleic acids. The “protein-only” hypothesis for their inheritance led these elements to be called “yeast prions” (Wickner, 1994). The term “prion” was first coined to describe the infectious agent hypothesized to cause mammalian spongiform encephalogathies (TSEs) by a “protein only” mechanism: a normal cellular protein (PrPC) adopts an altered conformation (PrPSc) and interacts with other PrPC proteins to change their conformation as well (Prusiner, 1996).
The yeast [PSI+] element, the subject of the inventor's work, does not generally kill cells. It reduces the fidelity of ribosome translation termination and thereby suppresses nonsense codons (Lindquist, 1997). This phenotype is thought to result from a change in the state of the translation-termination factor, Sup35, that interferes with its normal function. In [psi−] cells, Sup35 is protease sensitive and is mostly soluble; in [PSI+] cells, Sup35 has increased protease resistance and is mostly aggregated (Paushkin et al., 1996; Patino et al., 1996; Paushkin et al., 1997). “Aggregate” is used in a general sense; Sup35 may be polymerized into an amyloid-like structure, or coalesced in a less ordered state. When pre-existing Sup35 is in the aggregated state, newly made Sup35 aggregates too, causing a self-perpetuating loss of function in the termination factor and a heritable change in translational fidelity (Patino et al., 1996; Paushkin et al., 1997).
[PSI+] depends upon the chaperone protein Hsp104. The first known function of Hsp104 was in thermotolerance in yeast, where it increases survival after exposure to extreme temperatures up to 1000-fold (Sanchez and Lindquist, 1990). It does so by promoting the reactivation of proteins that have been damaged by heat and have begun to aggregate (Parsell et al., 1994). At normal temperatures, Hsp104 overexpression cures cells of [PSI+]. Sup35 becomes soluble and the fidelity of translation termination is restored. This state is heritable, even when overexpression of Hsp104 ceases (Chernoff et al., 1995). Because the only known function of Hsp104 is to alter the conformational state of other proteins, these observations provide a strong argument that [PSI−1] is indeed based upon a heritable (self-perpetuating) change in the conformational state of Sup35.
Surprisingly, deletions of HSP104 also cure cells of [PSI+], and Sup35 is soluble in such cells as well (Patino et al., 1996; Chernoff et al., 1995). This is very different from heat-induced aggregates, which remain insoluble in hsp104 deletion strains. Clearly, the relationship between Hsp104 and [PSI−1] is more complex than the relationship between Hsp104 and thermotolerance.
1.2.2 Human Prions
The family of transmissible spongiform encephalopathies (TSEs) include scrapie in sheep, bovine spongiform encephalopathy (BSE) or “mad cow disease” in cattle, and several rare human neuropathies: Creutzfeld-Jacob disease (CJD), fatal familial insomnia (FFI), Gertsmann-Straussler-Scheinker (GSS) syndrome, and kuru (Caughey and Chesebro, 1997; Prusiner, 1996). A central event in TSE pathogenesis is the accumulation in the nervous system of an abnormally-folded version (PrPSc) of a normal cellular protein, PrPC. Griffith first proposed a “protein-only” model to explain the unconventional behavior of the infectious TSE agent (Griffith, 1967). Indeed, the “prion”, a term by which the agent is popularly known today, appears to be almost entirely proteinaceous: consisting primarily of PrPSc (Caughey and Chesebro, 1997; Prusiner, 1996).
Several lines of evidence show that PrPC is conformationally distinct from PrPSc although both molecules derive from the same primary sequence and have no detectable post-translational differences (Caughey and Chesebro, 1997; Prusiner, 1996; Caughey et al., 1991; Pan et al., 1993; Riek et al., 1996). The conversion of PrPC to PrPSc appears to involve direct interactions of PrPC with pre-existing PrPSc (Caughey and Chesebro, 1997; Prusiner, 1996; Kocisko et al., 1994). However, the exact mechanism underlying conversion is not known. Genetic and inhibitor studies have suggested that other cellular factors may influence TSE pathogenesis or serve as regulators of disease (Kenward et al., 1996; Talzelt et al., 1996; Carlson et al., 1988; Caughey et al., 1994; Telling et al., 1995; Edenhofer et al., 1996). None have been conclusively identified; however, cellular osmolytes (sometimes called chemical chaperones; Caughey and Raymond, 1991) and protein chaperones have been frequently speculated to be among them (Kenward et al., 1996; Caughey et al., 1994; Telling et al., 1995; Edenhofer et al., 1996).